【作者】 李莉；

【导师】 张诗武； 尹玉；

【作者基本信息】 安徽医科大学 ， 病理学与病理生理学， 2014， 硕士

【摘要】 目的：津白Ⅱ（Tientsin Albinao2，TA2）小鼠具有高自发性乳腺癌特点，是研究人乳腺癌的良好动物模型，本研究通过研究Wnt通路相关蛋白、小鼠乳腺肿瘤病毒（mousemammary tumor virus,MMTV）在TA2小鼠乳腺癌生成中的作用，阐述TA2小鼠自发性乳腺癌发生相关机制。方法：1.正常雌性成年处女TA2小鼠60只，鼠龄6~8周，体重20~25g，将小鼠去势后随机分成四组，联合给药组（CT组，给药剂量17β-雌二醇0.02mg+孕酮0.2mg溶解于0.1ml麻油中）、雌二醇组（E组，给药剂量17β-雌二醇0.02mg溶解于0.1ml麻油中）、孕酮组（P组，给药剂量孕酮0.2mg溶解于0.1ml麻油中）及对照组（C组，给予0.1ml纯麻油），给药频率1次/2周，10月后观察雌激素、孕激素对TA2小鼠乳腺肿瘤生成数量的影响，并分析雌激素、孕激素在TA2小鼠乳腺癌发生过程中的作用。2. Real-time PCR方法分别检测肿瘤及乳腺组织中MMTV-LTR mRNA、Wnt1mRNA、Wnt5a mRNA、Wnt5b mRNA、Wnt10b mRNA表达水平并分析其相关性。RT-PCR方法检测各组小鼠肿瘤及乳腺组织中MMTV-LTR mRNA表达。3.免疫组织化学技术检测TA2小鼠正常乳腺组织、自发性成瘤TA2小鼠乳腺癌前病变组织及肿瘤组织中Wnt1、Wnt5a、β-连环蛋白（β-catenin）的表达。进一步统计分析Wnt1、Wnt5a、β-catenin在TA2小鼠正常乳腺组、自发性成瘤TA2小鼠乳腺癌前病变组及肿瘤组中肿瘤阳性细胞表达率，并分别比较差异性。结果：1.15只联合给药组小鼠中有10只在连续给药10月后发瘤（肿瘤发生率66.7%），而其他三组无一例发瘤（肿瘤发生率0%）。联合给药组中，10只小鼠的肿瘤均为单发，5个小鼠肿瘤定位于左下腹，3个小鼠肿瘤定位于右下腹，两个小鼠肿瘤位于颈部。联合给药组小鼠乳腺癌细胞呈巢状、实体和腺管状排列，细胞核分裂象多见；联合给药组小鼠乳腺导管不规则，有些导管形成囊状结构，乳腺导管比正常乳腺导管大；雌激素组、和孕激素组小鼠的乳腺导管规则，导管上皮细胞形态没有明显异常；而对照组小鼠乳腺导管萎缩，为脂肪组织所替代。2. Real-time PCR结果显示：Wnt1mRNA、Wnt5a mRNA、Wnt5b mRNA、Wnt10bmRNA在联合给药组小鼠乳腺组织表达水平最高，同时对照组小鼠乳腺上皮细胞中表达水平最低，两组之间分别比较，差异有统计学意义（P=0.000）。较其他组比较，联合给药组小鼠肿瘤及乳腺组织中MMTV-LTR mRNA表达量较高，差异有统计学意义（P=0.000）。3.免疫组织化学检测结果提示：Wnt1、Wnt5a在小鼠正常乳腺组织中阴性表达，在自发性成瘤小鼠乳腺癌前病变和乳腺癌组织细胞质表达且在自发性成瘤小鼠乳腺癌前病变的表达强度高于其在乳腺癌组织中表达。β-catenin在正常小鼠乳腺组织主要在细胞膜上表达，在自发性成瘤TA2小鼠乳腺癌前病变组织及肿瘤组织胞质中均有异常表达，且在肿瘤组织的表达强度高于其在癌前病变组织中的表达。Wnt1、Wnt5a、β-catenin在自发性成瘤小鼠乳腺癌前病变组、肿瘤组中的阳性细胞表达率显著高于正常乳腺组，差异有统计学意义（P=0.000）。Wnt1、Wnt5a在小鼠乳腺癌前病变组细胞阳性率高于肿瘤组，其中Wnt1差异有统计学意义（P=0.001），Wnt5a差异无统计学意义（P=0.239）。β-catenin在肿瘤组的肿瘤细胞阳性率明显高于乳腺癌前病变组，差异有统计学意义（P=0.005）。结论：1.在构建的TA2小鼠动物模型中，雌孕激素联合给药促进TA2小鼠乳腺肿瘤形成。MMTV及Wnt信号通路相关蛋白在TA2小鼠乳腺癌组织中表达异常，可能在TA2小鼠乳腺癌形成过程中发挥重要作用。2.在TA2小鼠自发性乳腺癌组织中，Wnt信号通路相关蛋白表达异常，提示它们在TA2小鼠自发性乳腺癌形成过程中发挥重要作用。更多还原

【Abstract】 Objective: TA2mice had a high incidence of spontaneous breast cancer.There weregood models for researching the mechanism of breast cancer.In order to clarify themechanism of the high incidence of spontaneous breast cancer,we studied the role of theWnt signal related proteins,MMTV in the development of breast cancer in TA2mice.Methods:1. Sixty virgin female TA2mice which weigh from20to25g were equally divided intofour groups randomly, the estradiol group(E group, received0.1ml of0.02mg of17β-estradiol dissolved in sesame oil),the progesterone group(P group, received0.2mgof progesterone dissolved in sesame oil), the combination treatment group(CTgroup,received0.1ml sesame oil containing0.02mg17β-estradiol and0.2mgprogesterone) and the control group（C group,received0.1ml sesame oil alone）.Allthese mice of the four groups were injected twice a week.Then compared the occurrenceof breast cancer in the four groups after10months and analyzed the correlation of them.2. Real-time PCR was employed to determine the relative expression level ofMMTV-LTR mRNA、Wnt1mRNA、Wnt5a mRNA、Wnt5b mRNA、Wnt10b mRNAin the breast cancer and mammary gland，then analyzed the correlation of them.RT-PCR was used to detect the MMTV-LTR mRNA sequence of both the mammarygland tissues and the tumor tissues collected from each group.3. Mammary tissue samples from normal TA2mice, precancerous lesions and cancertissues of spontaneous breast cancer-bearing TA2mice were subsequently used to detectthe expression of Wnt1,Wnt5a and β-catenin via immunohistochemical staining.Then Compared the positive expression rate of Wnt1,Wnt5a and β-catenin in normal breastgroup,precancerous group and breast cancer group,respectively.Analyzed the correlationof them,respectively.Results:1.10out of15mice in the CT group developed breast cancer(the incidence was66.7%),but no mouse in the other three groups did(the incidence was0%).In the CT group, eachmouse with cancer developed only one tumor mass. Five mice had tumors located in thelower left abdomen, three mice had tumors located in the lower right abdomen and twomice had tumors located in the neck.The breast cancer cells were formed solid nests orglandular patterns.There were numerous cells undergoing mitosis with less stroma thanfound in normal mice.Also in these mice,Mammary glands had an irregular shape, andsome were cystoid. The size of the glands was also larger than that of normal mice.Inthe E and P groups, the morphological change of the mammary ductal structure wasintegrated and regularly shaped, and the epithelial cells appeared normal.In the C group,the mammary gland ducts were atrophied and replaced with fat tissue.2. Mammary tissue from the CT group had the highest expression of Wnt1, Wnt5a,Wnt5b and Wnt10b mRNA, while mammary epithelium from the C group had thelowest level of expression. The differences among the groups were statisticallysignificant,respectively,(P=0.000).The tumor tissue and mammary tissue samples fromthe CT group had higher levels of MMTV-LTR mRNA expression than did the othergroups,and the differences had statistically significant（P=0.000）.3. Results of immunohistochemical staining showed that the positive expression ofWnt1and Wnt5a located in cytoplasm in spontaneous breast cancers and mammarygland precancerous lesions from TA2mice. The normal mammary glands were negativefor Wnt1and Wnt5a staining. The cytoplasm of precancerous lesions showed higherexpression for Wnt1and Wnt5a than them in the breast cancer. β-catenin in breastcancer and precancerous lesions were abnormal cytoplasmic expression. The expression level in breast cancer was higher than in the precancerous lesions. In normal mammarygland, the positive expression of β-catenin mainly located in the cytomembrane. Thepositive expression rates of Wnt1,Wnt5a,β-catenin in precancerous group and breastcancer group were significantly higher than those in normal breast group (P=0.000).Thepositive expression rates of Wnt1,Wnt5a in precancerous group were higher than in thetumor group,and the differences of Wnt1had statistical significance (P=0.001) whileWnt5a did not (P=0.239). Breast cancer group had higher positive expression rate ofβ-catenin than the precancerous group and the differences also had statisticalsignificance (P=0.005).Conclusion:1. Combined treatment with estradiol and progesterone promote the formation of breastcancer in TA2mice.MMTV and Wnt signal pathway related proteins were abnormallyexpression in the breast cancer in TA2mice, they may play important roles in thedevelopment of breast cancer in TA2mice.2. Wnt signal pathway related proteins which expression abnormally in the spontaneousbreast cancer may play important roles in the development of spontaneous breast cancerin TA2mice.更多还原